68Ga-cyc-DX600 was prepared in house with DOTA-modified cyclic DX600 peptide (cyc-DX600-DOTA, DX600 peptide with condensed disulfide bond of cysteine) as the precursor following the reported protocol [11 (link), 12 ]. In detail, the newly eluted 68Ga3+ in 4 mL 0.05 M HCl was mixed with precursor in 1 mL 0.25 M NH4Ac, heated to 100 ℃ and maintained for 10 min. Radiochemical purity (RCP) was measured with HPLC system (1260 Infinity, Agilent Technologies) equipped with a radioactive detector (Flow-count, Eckert & Ziegler) to determine the labeling rate, as well as the stability in PBS and 5% fetal bovine serum for one hour incubation at room temperature.
18F-FDG was purchased from Atom Kexing Radiopharmaceuticals Ltd with strict quality control. Radiopharmaceuticals with labeling rate higher than 95% were used in imaging research immediately, and the specific radioactivity of 68Ga-cyc-DX600 was controlled as about 3.7 MBq/µg in the imaging research on various tumor-bearing mice models or patients.
To verify the maintain of ACE2 targeting ability on cellular level, 125I-labeled DX600 and 125I-labeled cyc-DX600-DOTA were synthesized via Iodogen-catalyzed iodization with I-125. Cellular binding was performed on 1 × 105 HEK-293T/hACE2 cells that expressed humanized ACE2 protein. After the co-culture with 1 nmol radiopharmaceuticals for 30 min or 1 h, the excess radiopharmaceuticals were washed away by cold PBS for three times, and the bound peptide or precursor was quantified with a gamma-detector.
18F-FDG was purchased from Atom Kexing Radiopharmaceuticals Ltd with strict quality control. Radiopharmaceuticals with labeling rate higher than 95% were used in imaging research immediately, and the specific radioactivity of 68Ga-cyc-DX600 was controlled as about 3.7 MBq/µg in the imaging research on various tumor-bearing mice models or patients.
To verify the maintain of ACE2 targeting ability on cellular level, 125I-labeled DX600 and 125I-labeled cyc-DX600-DOTA were synthesized via Iodogen-catalyzed iodization with I-125. Cellular binding was performed on 1 × 105 HEK-293T/hACE2 cells that expressed humanized ACE2 protein. After the co-culture with 1 nmol radiopharmaceuticals for 30 min or 1 h, the excess radiopharmaceuticals were washed away by cold PBS for three times, and the bound peptide or precursor was quantified with a gamma-detector.
